GSM6502515: zebrafish, Ribo-seq, Eif4e1c -/-, rep 1 [r_mR1]; Danio rerio; OTHER
The hearts were lysed on ice with 100ul of lysis buffer with 53 mg/ml cycloheximide using mechanical homogenization with a pestle for thirty seconds. Cells were lysed further by trituration of the sample using a 27.5-gauge needle syringe 20x on ice. Cell extracts (average OD 7) were treated with MNase (NEB), CaCl2 (5mM final concentration), and Turbo DNAse I (Invitrogen) at 25°C for 30 minutes. MNase digestion was stopped with SUPERase-inhibitor incubation on ice. 80S ribosomes were isolated using SH400 spin columns (Cytiva). Columns were prepared according to the manufacturer's instructions. 100 µls of MNase treated sample was loaded per column and centrifuged for 2 min at 600 RCF. The flow through was isolated and incubated for 5 min. with 3x volume of Trizol LS (Ambion). 200 µl of chloroform was added and incubated for 2 min. Samples were centrifuged for 15 min. at 12,000 rpm (4°C). The aqueous phase was extracted and precipitated overnight at -20°C using glycoblue (ThermoFisher) and isopropanol followed by centrifugation for 30 minutes at 4C at 10,000 RPM. Pellets were washed with 75% Ethanol and resuspended into RNase free water. Next-Flex smRNA seq kit v3